Mitochondria as the major source of energy generation are essential for proper cellular function. There is considerable evidence supporting the key role of mitochondrial dysfunction in heart disease such as myocardial infarction and heart failure. At the myocardial level of the post-ischemic heart, a defect in energy metabolism associated with overproducing oxygen free radicals and NO in mitochondria was marked. Alterations of protein S-glutathionylation (PrSSG) and protein nitration have been detected in the mitochondrial complex I (NQR), complex II (SQR), and other ETC proteins during myocardial ischemia and reperfusion injury. Alterations of NQR/SQR-derived oxidative modifications are closely linked to oxygen free radical production, NO metabolism, and homeostasis of redox thiols in mitochondria. S-glutathionylation of the reactive and labile cysteine residues in the NQR or SQR is a reversible modification, whereas S-sulfonation of reactive cysteine residues is an irreversible modification. Our central hypotheses are that both cysteinyl modifications are highly regulated by the redox status in the mitochondria of the post-ischemic heart, and the mitochondrial redox status is controlled by ROS production, NO metabolism, and the homeostasis of the GSH pool. The long term objectives of this research are to elucidate the molecular mechanism of mitochondrial redox signals in the mediation of myocardial injury, to understand the pathogenesis, and to develop a treatment for cardiovascular diseases. The key hypotheses of the major signal pathway leading to protein S-glutathionylation/sulfonation in mitochondria will be tested by pursuing the following specific aims using novel animal models, EPR spectrometry, and mass spectrometry. Specific aim 1 will determine whether irreversible protein S- sulfonation of NQR and SQR is induced in the mitochondria of the post-ischemic myocardium. The protein sulfonation marked in the NQR and SQR after myocardial infarction will be characterized by mass spectrometry. A sequence-specific antibody for sulfonation will be generated to detect this event in vitro and in vivo. EPR spectrometry with a spin probe and a spin trap will be used to measure the redox status and O2- generation activity of mitochondria isolated from the post-ischemic heart. Specific aim 2 will determine the role of glutathione reductase (GR) in the mechanism of glutathionylation of NQR/SQR and regulation of overall mitochondrial function in the post-ischemic heart. We will use a pharmacologic approach and mice deficient in GR (gsr-/-) to determine whether (i) enhancing GSSG in vivo will increase glutathionylation of NQR and SQR, and (ii) whether or not increasing NQR/SQR glutathionylation in vivo will be protective and reduce the susceptibility of mitochondria to post-ischemic injury. Specific aim 3 will ascertain the role of eNOS in the mechanism of NQR/SQR glutathionylation and regulation of overall mitochondrial function in the post-ischemic heart. Mice with an eNOS-/- and a cardiac-specific eNOS-/- genotype will serve as an excellent in vivo model for studying the regulation of NQR/SQR glutathionylation, mitochondrial redox status, and its O2- generation activity via NO metabolism. We will also create a novel mouse model by crossing cardiac-specific SOD2 transgenic mice with eNOS-/- mice in order to determine whether increased SOD2 signaling in mitochondria is sufficient to correct oxidative injury resulting from eNOS deficiency and post-ischemic injury.